Jiro Mitobe1, Fumiko Nishiumi2, Itaru Yanagihara2, Shouji Yamamoto1, Makoto Ohnishi1
- Department of Bacteriology I, National Institute of Infectious Diseases, Shinjuku, Tokyo, Japan
- Department of Developmental Medicine, Research Institute, Osaka Women’s and Children’s Hospital, Izumi, Osaka, Japan
The rod shape of bacilli is maintained by bacterial cytoskeletal protein MreB, an actin homolog that acts in concert with the inner membrane protein RodZ. We previously reported RodZ binds RNA to control the posttranscriptional regulation of invE (virB), which controls the type III secretion system essential for the virulence of Shigella. Here, we show that purified RodZ forms “superstructures” of high molecular mass that dissociate into a midsized “basal complex” in the presence of nonionic detergent, or to a monomer in the presence of dithiothreitol. We used mass spectrometry to show that the basal complex was a hexamer. Electrophoresis mobility shift assays combined with gel filtration detected the RNA-binding activity in fractions containing molecules larger than the basal hexamer. The superstructure was consistently detected with MreB in crude cell lysates of S. sonnei that were fractionated using gel filtration. Immunofluorescence microscopy using two different super-resolution settings showed that wild-type RodZ was distributed in cells as separate dots. Consistent with the superstructure comprising homohexamers, majority of the dots distributed among areas of discrete values. In addition, simultaneous immunodetection of MreB provided the first evidence of colocalization with RodZ as larger patch like signals. These findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.
CovalX Technology Used
In this research, the dissociation of “superstructures” of high molecular mass, formed from purified RodZ, into a midsized “basal complex” (in the presence of nonionic detergent) or to a monomer (in the presence of dithiothreitol) is discussed.
The molecular mass of the complex containing RodZ was determined using CovalX AG Laboratory Analytical Service. The 9 μl sample for High-Mass MALDI MS contained ten serial dilutions (1 mg/ml–19.53 μg/ml) prepared in 10 μl PBS and an aliquot (1 μl), and was treated with CovalX K200 Stabilizer reagent (1 μl, 2 mg/ml). The sample was later analyzed using the CovalX HM3 interaction module (calibrated with BSA and IgG) to find species whose mass ranges from 0 kDa to 1500 kDa. CovaX’s Complex Tracker software (ver. 2.0) was also used to process MS data to correct the mass of the crosslinker.
The mass spectrometry was used to show that the basal complex was a hexamer, and the findings indicate that native RodZ forms clusters of various sizes, which may correspond to a superstructure comprising multiple hexamers required for the RNA-binding activity.